1. Field of the Invention
The invention is in the field of antibody-based assays for toxins having peptidase activity. In particular, this invention relates to assays for toxins, in particular botulinum neurotoxins and tetanus toxins. The invention also relates to antibodies useful in the assays and to peptides immobilized on solid phase supports that are useful in the assays.
2. Related Art
The botulinum neurotoxins are a family of structurally similar, but antigenically different protein neurotoxins which act on the peripheral nervous system to block neuromuscular transmission. These neurotoxins are extremely potent, with a human lethal dose in the order of micrograms, and give rise to the rare but frequently fatal disease, botulism. Assays for the botulinum neurotoxins are currently used in both the food and pharmaceutical industry. The food industry employs assays for the botulinum neurotoxins to validate new food packaging methods and to ensure food safety. With the growing clinical use of the botulinum toxins, the pharmaceutical industry requires accurate assays for these toxins for both product formulation and quality control.
It is known to assay for botulinum toxin in foodstuffs using the mouse lethality test. This test has been the industry standard for many years, though over the past 10 years a number of immunoassay methods have been developed in an attempt to replace the mouse test in the majority of applications.
One such assay operates by addition of a test sample to a plate or column to which is attached an antibody that binds to toxin present in the sample. A further antibody is typically used to detect bound toxin. These enzyme-linked immunoassays (ELISA) have the advantages that they are specific to one botulinum toxin type and can be performed rapidly, in less than 2 hours. The ELISAs, however, suffer from several drawbacks:
(a) They do not measure the biological activity of the toxins, PA1 (b) They cannot distinguish between active and inactive toxin, and PA1 (c) Due to antigenic variations some toxins are not detected by these assays which therefore give rise to false negatives. PA1 (a) It is expensive and uses large numbers of laboratory animals, PA1 (b) It is non-specific unless performed in parallel with toxin neutralization tests using specific anti-sera, and PA1 (c) It is not very accurate unless large animal groups are used. PA1 (a) combining a test compound with a substrate and with antibody, wherein the substrate has a cleavage site for the toxin and when cleaved by toxin forms a product, and wherein the antibody binds to the product but not to the substrate; and PA1 (b) testing for the presence of antibody bound to the product, which product is attached to a solid phase assay component. PA1 (a) a peptide linked to a solid-phase, the peptide being cleavable by the toxin to generate a cleavage product, PA1 (b) an antibody that binds to the cleavage product but not to the peptide, and the assay may comprise the steps of: PA1 (i) combining the test compound with a solid phase comprising a peptide selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof, PA1 (ii) washing the test compound from the solid phase, PA1 (iii) combining the solid phase with an antibody adapted for binding selectively with peptide cleaved by toxin, and PA1 (iv) detecting a conjugate of the antibody with cleaved peptide. PA1 (i) adding a test solution to an assay plate comprising immobilized peptide, the peptide being selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof, PA1 (ii) incubating the assay plate, PA1 (iii) washing the plate with a buffer, PA1 (iv) adding to the plate an antibody solution, said solution comprising an antibody adapted selectively to bind to a peptide selected from the group consisting of(l) a peptide the C-terminal end of which is selected from the group consisting of SEQ ID NOS: 1, 3 and 5, and (2) a peptide the N-terminal end of which is selected from the group consisting of SEQ ID NOS: 2, 4 and 6, PA1 (v) incubating the assay plate, PA1 (vi) washing the plate with a buffer, and PA1 (vii) measuring the presence of antibody on the assay plate. PA1 (i) identifying a macromolecule that is cleaved by the toxin, thereby forming at least first and second cleavage products, PA1 (ii) immunizing an animal with a selected one of the cleavage products, PA1 (iii) isolating from the animal an antibody that binds to the selected cleavage product, and PA1 (iv) checking the antibody does not bind to the macromolecule. PA1 (i) immunizing an animal with an antigen selected from the group consisting of (1) SEQ ID NO.s 1-6; and (2) a carrier molecule linked to (1), and PA1 (ii) isolating from the animal an antibody that binds to the antigen. PA1 (1) an assay component according to the invention; PA1 (2) (a) an antibody according to the invention which is linked to or including an enzyme, or PA1 (b) an antibody according to the invention which is unlabeled with an enzyme and a second antibody adapted to bind to the first antibody and linked to or including an enzyme, and PA1 (3) a substrate for the enzyme.
The botulinum neurotoxins have recently been shown to possess highly specific zinc-endopeptidase activities within their light sub-units. Depending on the neurotoxin type these act to cleave small proteins within the nerve cell which are involved in neurotransmitter release. Botulinum types A and E toxins cleave protein SNAP-25. Botulinum types B, D, F and G and tetanus toxins cleave vesicle-associated membrane protein (VAMP-also called synaptobrevin). Botulinum type C toxin cleaves the protein syntaxin.
In the development of further toxin assays, various procedures have been devised for the evaluation of endopeptidase activities. Liquid chromatography procedures are known and are based on resolution of the peptide product and subsequent evaluation. These procedures are time-consuming, expensive and do not lend themselves readily to automation. It is also known to use spectrophotometric methods, requiring the development of suitable chromogenic peptide reagents. Such methods provide a continuous precise assay for endopeptidases. Spectrophotometric methods, however, require relatively pure preparations of enzyme and are not normally suitable for evaluation of endopeptidase activities in crude or particulate samples.
Despite these efforts, at present, the only convenient assay for the biological activity of the botulinum neurotoxins, and the only assay that is FDA approved, remains the mouse lethality test. This test suffers from a number of drawbacks:
The present invention describes a novel assay system for toxins, using novel reagents. The assay aims to overcome or at least mitigate many of the drawbacks of present in vitro assays for these toxins.